| Project/Area Number |
10670260
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Bacteriology (including Mycology)
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| Research Institution | Kagawa Medical University |
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Principal Investigator |
KATAYAMA Seiichi Kagawa Medical University, Medicine. Research Associate, 医学部, 助手 (70169473)
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| Co-Investigator(Kenkyū-buntansha) |
MATSUSHITA Osamu Kagawa Medical University, Medicine. Associate Professor, 医学部, 助教授 (00209537)
OKABE Akinobu Kagawa Medical University, Medicine. Professor, 医学部, 教授 (20093677)
南 純三朗 香川医科大学, 医学部, 助教授 (40157566)
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 1999: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1998: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Keywords | Clostridium perfringens / phospholipase C / transcriptional regulation / phased A-tracts / RNA polymerase / RNAポリメラーゼ / UPエレメント |
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| Research Abstract |
The Clostridium perfringens phospholipase C gene (plc) possesses three phased A-tracts upstream of the promoter (-66 to 40). The A-tracts form bent DNA, facilitate the formation of the RNA polymerase (RNAP)-plc promoter complex through extension of the contact region, and activate the transcription in a low temperature-dependent manner. In order to clarify the mechanism of the transcriptional regulation by the A-tracts, it seems important to elucidate specific subunit/domain of RNAP and the A-tracts responsible for the extended contact.
We cloned rpoA encoding α subunit of the C. perfringens RNAP. The deduced amino acid sequence was identical to the N-terminal sequence of purified RNAP α subunit. The coding region of rpoA was cloned into an expression vector to overproduce and purify the α subunit (α-WT, 315 aa). N-terminal (α-NTD, 228 aa) and C-terminal (α-CTD, 79 aa) domains of the subunit were also purified in the same manner.
The gel retardation assays showed that α-WT and α-CTD bind to a DNA fragment containing three phased A-tracts and plc promoter (3Ap), but that α-NTD does not. From this result, a possibility was raised that α-CTD binds to the A-tracts. We are trying to show the contact region between 3Ap and α-CTD by hydroxyl radical footprinting at the moment.
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